This invention is related to the tissue-affinitive collagen used as carrier of bone morphogenetic protein and the method of producing the same.
Hitherto, when bone defect in a living body was to be filled, it was usual that autogeneous bone was removed from patient's own bone to be used for autogeneous bone grafts. This is because the process of incorporation of autogeneous bone grafts is earlier and more reliable than those of homogeneous or heterogeneous bone grafts. However, there are some such disadvantages to the use of autogeneous bone grafts as (1) the amounts of removed autogeneous bone is naturally limited, (2) removing autogeneous bone requires an additional surgical procedure on the same patient, heightening the risk of infection, extending the operating time, and leading to pain on the donor site. Therefore, when the bone defect is large, artificial biomaterials such as ceramics and metals have been used for bone graft substitutes. Such artificial biomaterials, however, can not readily be incorporated into surrounding bone tissue, because sufficient amounts of bone are not formed around the biomaterials.
To settle this problem, we have devised the use of osteoinductive biomaterials comprising a complex of collagen and bone morphogenetic protein or a composite of collagen, bone morphogenetic protein and tissue affinitive materials such as ceramics with a desired shape for filling and fixation of bone defects (J. P., A., No. 60-253455, U.S. Ser. No.737,386).
Both collagen and bone morphogenic protein (BMP) are known in the art and in the related literature Collagen has been well-established in the aspects of the chemistry, molecular structure, biochemical properties, and immunology, and enzyme-solubilized collagen is usually used as biomaterial because of the relatively low antigenicity (for example, U.S. Pat. No. 4,314,380). BMP was separated from Dunn osteosarcoma by Takaoka et al. (Biomedical Research 2: 466-471, 1981) and from bone tissue by Urist (U.S. Pat. No. 4,294,753). Jefferies (U.S. Pat. No. 4,472,840) demonstrated the method of inducing osseous formation by implanting bone graft material, which was prepared from both collagen and demineralized bone particles and/or bone morphogenic protein derived from bone.
We have prepared osteoinductive biomaterials using BMP which was separated from Dunn osteosarcoma and purified as described by Takaoka et al., and collagen, which was enzyme-solubilized collagen prepared by a conventional method, to study the osteogenic activity by implantation of the biomaterials into dorsal muscles of mice and found the following: On sole implantation of BMP, a large quantity of BMP, was required to induce ectopic bone formation, which was consistently observed when much less quantity of BMP was implanted in combination with collagen.
However, when enzyme-solubilized collagen prepared by a conventional method was used, the ectopic bone formation was not always induced smoothly, and the achievement was remarkably dependent on the amount of telopeptides in collagen used, which is closely related to the antigenecity of the collagen. Thus, when collagen containing less amount of telopeptides was used, the quantity of bone induced for a definite quantity of BMP implanted was increased, the minimum quantity of BMP required to induce ectopic bone formation was decreased, and higher reproducibility in the experiment was obtained. That is, collagen with lower antigenicity is required for more satisfactory induction of ectopic bone formation using BMP, and because of low yield of BMP when purified, the use of the collagen as carrier of BMP is also essential for the practical use.
Since the site of antigen of collagen exists mainly in non-helical terminal regions (telopeptides) of collagen molecule, a procedure to eliminate antigenicity has been taken through removal of the telopeptides of collagen molecule by allowing proteolytic enzymes, such as pepsin to act on collagen (for example, Int. Rev. Conn. Tiss. Res. 7. 61-99 (1976)).